Solution for dissociating vitamin D from vitamin D-binding protein, associated detection method and use

ABSTRACT

At least one fluoroalkyl surfactant and of at least one alcohol having 1 to 4 carbon atoms are used for dissociating vitamin D and/or a vitamin D metabolite from vitamin D binding protein. A solution comprising the at least one fluoroalkyl surfactant and at least one alcohol is provided. A method for detecting and quantifying, in vitro, vitamin D and/or at least one vitamin D metabolite in a biological sample includes treating the sample by incorporating at least one fluoroalkyl surfactant and at least one alcohol having 1 to 4 carbon atoms, so as to dissociate the vitamin D and/or its metabolite(s) to be detected from vitamin D binding protein; and detecting and quantifying vitamin D and/or at least one of its metabolites, in particular by immunoassay. A kit for detecting and quantifying vitamin D and/or at least one vitamin D metabolite by immunoassay and including the solution is provided.

The present invention relates to the technical field of detectingvitamin D. More particularly, the invention provides the use of anassociation of a fluoalkyl surfactant and an alcohol for releasingvitamin D and/or one of its metabolites from the Vitamin D bindingprotein, a solution containing such an association, an in vitro methodof detecting/quantifying vitamin D and/or at least one metabolite ofvitamin D using such an association, and a kit for detecting/quantifyingby immunoassay making use of such an association.

Vitamin D is an important substance having numerous implications in thebiological processes of the human and animal body. Biologically activevitamin D is known to regulate, amongst other things, the fixing ofcalcium from the intestine, and bone mineralization, and it has aninfluence on many other metabolic pathways, such as for example theinsulin system. A deficiency or an excess of vitamin D can have variousconsequences. In particular, it is known that vitamin D deficiency leadsto severe illnesses such as osteoporosis and rickets.

Furthermore, excess vitamin D, in particular due to an overdose, istoxic. In particular, a high level of vitamin D can lead tohypercalcemia caused by an increase in the absorption of calcium by theintestine. Other toxic effects of vitamin D are manifested by anincrease in blood pressure, gastrointestinal troubles such as anorexia,nausea, often followed by excessive production of urine, polydipsia,fatigue, nervousness, itching, or indeed kidney failure.

As mentioned in application WO 2012/091569, it has also been discoveredin recent years that vitamin D modulates the immune system and reducesinflammation. It has also been suggested that vitamin D can preventcancers of the colon, the ovaries, and the breast.

Consequently, it is important to be able to measure or quantify vitaminD, i.e. to determine its concentration, so as to reveal any potentialdeficiencies or excesses.

Vitamin D is present in the organism in two forms, namely vitamin D₂(ergocalciferol) and vitamin D₃ (cholecalciferol) of formulae that areset out below (in which the positions in the vitamin D are numbered incompliance with steroid nomenclature).

Vitamin D₂ is the exogenous form of vitamin D, coming from food. VitaminD₃ is the endogenous form of vitamin D as produced by the organism underthe action of ultraviolet rays from sunlight on the skin. The vitamin D₃that is produced by the skin binds with the vitamin D binding protein,which transports it to the liver. Both forms may also come fromnutritional supplements. Various metabolites of vitamin D are produced.In particular, two-step metabolization takes place, the first stepconsisting in producing 25-hydroxy vitamin D (D₂ or D₃), followed byproducing 1,25-dihydroxy vitamin D (D₂ or D₃).

Measuring the quantity of vitamin D itself is of limited interest, giventhat its concentration fluctuates strongly as a function of diet. Thesame applies to 1,25-dihydroxy vitamin D metabolites which are presentin fairly low concentration and which also fluctuate.

The circulating forms of vitamin D are essentially metabolites of the25-hydroxy vitamin D type. Thus, the preferred mode for obtaininginformation about the overall concentration of vitamin D in a patient isassaying 25-hydroxy vitamin D.

The bonding of 25-hydroxy vitamin D, or more generally of vitamin D andits metabolites, to the vitamin D binding protein (DBP) complicatesassaying its components. In order to obtain an adequate assay, it isnecessary to release the hapten to be assayed from the DBP by causingthem to dissociate. Thus, various solutions have been proposed forobtaining the release of vitamin D and its metabolites afterdissociating DBP and before detecting them.

Various techniques listed in patent application WO 2007/039194 and inpatent application WO 2012/091569 have been developed. Only some of themare listed below.

An old technique, used in particular in application WO 99/67211 consistsin preparing a sample of plasma or serum in order to determine vitamin Dby ethanol precipitation. The precipitates are then eliminated in orderto recover the ethanolic supernatant containing the soluble metabolitesof vitamin D. Precipitation by means of alcohol or another organicsolvent such as acetonitrile has commonly been used in the past.Nevertheless, that technique cannot be automated and requires numerousmanual operations (adding solvents to the serum, mixing, centrifuging,recovering the organic phase, drying on a column or otherwise,re-suspension in a liquid solvent), thus making it obsolete these daysbecause of the high degree of variation that is observed betweenoperators.

Patent application WO 2007/039194 proposes using a solution containing5% to 30% by volume of one or more amphiphilic reagents selected fromdimethylsulfoxide (DMSO) and liquid organic amides, and optionally 0.7%to 8% by volume of a short chain alcohol (C₁-C₃). The amphiphiliccompounds used in that document are substances that are toxic, or indeeddangerous if it is DMSO.

Patent applications WO 2011/122948 and WO 2012/091569 in the name ofFuture Diagnostics describe immunoassay and an agent that enable vitaminD to be released from its binding protein and that uses a fluoroalkylsurfactant. The Applicant has made use of that solution, but hasnevertheless found that the dissociation that is obtained is still notsufficient.

The object of the invention is to propose a more effective dissociationtechnique that is easy to perform and that leads to effective release ofvitamin D and its metabolites, after dissociating the vitamin D bindingprotein (DEP), subsequently enabling them to be detected and quantifiedadequately.

In the context of the present invention, the object is to provide a newsolution for dissociating vitamin D or one of its metabolites fromvitamin D binding protein (DBP), that is more effective than thetechniques proposed in the prior art by Future Diagnostics in its patentapplications WO 2011/122948 and WO 2012/091569. In addition, thesolution described in the present invention does not present thetoxicity problems of the solutions as proposed in particular in patentapplication WO 2007/039194.

In this context, the invention provides the use of at least onefluoroalkyl surfactant, and in particular a perfluoroalkyl surfactant,and of at least one alcohol having 1 to 4 carbon atoms for dissociatingvitamin D and/or a vitamin D metabolite from vitamin D binding protein.Such joint use makes it possible to increase significantly thedissociation rate of vitamin D or one of its metabolites from vitamin Dbinding protein, in comparison with a similar use differing solely bythe absence of alcohol. In the use of the invention, the fluoroalkylsurfactant, and in particular the perfluoroalkyl surfactant, and thealcohol having 1 to 4 carbon atoms are incorporated in a liquid sampleincluding the vitamin D and/or a vitamin D metabolite (referred to as ananalyte or vitamin D analyte), associated with vitamin D bindingprotein. The analyte for dissociation is thus in contact both with thefluoroalkyl surfactant and with the alcohol, which, when used together,enable dissociation to be achieved that is greater than when using thesame fluoroalkyl surfactant on its own. The joint use of fluoroalkylsurfactant, and in particular perfluoroalkyl surfactant, and alcoholhaving 1 to 4 carbon atoms, leads to effective dissociation betweenvitamin D binding protein and vitamin D and/or one of its metabolites.

In particular, the fluoroalkyl surfactant and the alcohol are used inquantities such that the ratio multiplied by 100 of the weight ofsurfactant, when it is solid, or of the volume of surfactant, when it isliquid, over the volume of alcohol lies in the range 10% to 60%,preferably in the range 15% to 40%, and more preferably in the range 15%to 300. The solid or liquid nature is assessed at ambient temperature(in particular at 22° C.) and at atmospheric pressure (in particular at1013 hectopascals (hPa)).

Advantageously, the fluoroalkyl surfactant is selected fromperfluorocarboxylic acids, perfluorosulfonic acids and their salts, andin particular from perfluorohexanoic acid, perfluoroheptanoic acid, andperfluorooctanoic acid, and their salts. Perfluorohexanoic acid is alsoknown as perfluorocaproic acid or indeed as undecafluorohexaonic acid;its chemical abstract service (CAS) registration number is 307-24-4.Perfluorooctanoic acid is also known as perfluorocaprylic acid or indeedas pentadecafluorooctanoic acid; its CAS number is 335-67-1. In general,the salts of these perfluoroalkyl acids are solid, whereas thecorresponding free acids are liquid.

Perfluorohexanoic acid, possibly in salt form, is the preferredfluoroalkyl surfactant, since it presents better degradability comparedwith longer chain fluoroalkyl surfactants.

In preferred manner, the alcohol used has 1 to 3 carbon atoms and isselected from methanol, ethanol, n-propanol, and isopropanol. Methanolis the preferred alcohol in the invention since it makes it possible toobtain performance that is better in terms of the reproducibility of theresults of the assaying performed after dissociation, and provides agood compromise in terms of improving dissociation and thereproducibility of the results obtained.

In particularly advantageous manner, the dissociation is performed withperfluorohexanoic acid and methanol.

In the context of the invention, the dissociation may be performed inthe presence of an additional surfactant selected, in particular fromblock copolymers based on ethylene oxide and propylene oxide,polysorbates, and polyethylene glycol ethers.

By way of example, the invention is performed to dissociate 25-hydroxyvitamin D, and in particular 25-hydroxy vitamin D₂ and/or 25-hydroxyvitamin D₃ from vitamin D binding protein.

The alcohol and the fluoroalkyl surfactant may be incorporatedseparately in the sample in which it is desired to obtain dissociation,or they may be incorporated simultaneously. Under such circumstances, asingle solution is used referred to as a “dissociation” solution, inorder to minimize manipulations.

The invention also provides such solutions comprising at least onefluoroalkyl surfactant, and in particular a perfluoroalkyl surfactant,and at least one alcohol having 1 to 4 carbon atoms.

Such solutions may be said to be aqueous and they include a largequantity of water, which generally represents more than 80% by volumerelative to the total volume of the solution.

Advantageously, such a solution comprises a percentage of fluoroalkylsurfactant expressed by volume when the surfactant is liquid, or byweight when it is solid, relative to the total volume of the solutionlying in the range 0.1% to 3%, preferably in the range 1% to 2%. Inequally preferred manner, such a solution comprises a percentage byvolume of alcohol relative to the total volume of the solution lying inthe range 0.5% to 10%, and preferably in the range 2% to 7%.

The same preferences concerning the choice of the alcohol and of thefluoroalkyl surfactant and their relative quantities, stated withreference to the use, apply to the dissociation solutions of theinvention.

In an advantageous variant, the dissociation solutions of the inventionalso contain another surfactant selected from block copolymers based onoxide ethylene oxide and propylene oxide, polysorbates, and polyethyleneglycol ethers. Without seeking to be tied to any particularinterpretation of the results, such an additional surfactant can improvethe solubility of vitamin D or of its metabolite that is to be assayed.The use of such an additional surfactant, such as Pluronic® F-127, whichis a polyol corresponding to a block copolymer based on oxide ethyleneoxide and propylene oxide serves in particular to improve thereproducibility of the results. It is found that the final assay is morereliable. By way of non-limiting example, it is possible to usePluronic® F-127 at a concentration in the dissociation solution thatpreferably lies in the range 0.1% to 3% (by volume relative to the finalvolume of the dissociation solution).

In general, the dissociation solutions of the invention are buffered, inparticular to a pH lying in the range 6 to 8.

The buffers conventionally used in the field of diagnosis may beincorporated in the solution, so as to obtain a pH in the desired rangeand so as to stabilize it. By way of example, a phosphate-bufferedsaline (PBS) buffer or a tris buffer (tris-hydroxymethyl aminomethane)may be used.

A base may be incorporated in order to adjust the pH. Such a base may beany base that is conventionally used for this purpose, such as KOH,NaOH, LiOH, or Na₂HPO₄.

Such a dissociation solution may be prepared by mixing a buffer ofconcentration that preferably lies in the range 1 millimole (mM) to 500mM, a fluoroalkyl surfactant of concentration that preferably lies inthe range 0.1% to 3% (by weight or by volume, depending on the solid orliquid nature of the surfactant, relative to the final volume of thedissociation solution) and an alcohol of concentration that preferablylies in the range 0.5% to 10% (by volume relative to the final volume ofthe dissociation solution) in demineralized water. The pH of thedissociation solution is adjusted depending on the selected buffer, byadding acid or by adding base so as to obtain a value lying in the range4 to 8, and preferably around 7. When the dissociation solution containsan additional surfactant, it may be introduced at any stage.

Advantageously, such dissociation solutions do not contain any of thefollowing compounds: dimethyl sulfoxide, dimethylformamide,N,N-dimethylacetamide, tetramethylurea, N-methylpyrrolidone,1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone, triamide hexamethylphosphoric acid.

The invention also provides a detection and quantification method fordetecting and quantifying, in vitro, vitamin D and/or at least onevitamin D metabolite in a biological sample, the method comprising thefollowing steps:

a) a step of treating the sample by incorporating at least onefluoroalkyl surfactant and at least one alcohol having 1 to 4 carbonatoms, so as to dissociate including at least one fluoroalkyl surfactantand at least one alcohol having 1 to 4 carbon atoms from vitamin Dbinding protein; and

b) a step of detecting and quantifying vitamin D and/or at least one ofits metabolites.

The dissociation step a) should be performed before detection andquantification step b).

Advantageously, in such a detection and quantification method, thetreatment of step a) is performed by mixing the sample with adissociation solution of the invention. Usually, 1 to 20 volumes ofdissociation solution, preferably 5 to 10 volumes, more preferably 6 to8 volumes, and in particular about 7 volumes of solution are used for 1volume of sample. The selected volume is a function of the presumedconcentration of vitamin D and/or vitamin D metabolite that is to bedetected (referred to as analyte to be detected).

Advantageously, the detection and quantification method of the inventionis performed on a sample of blood, of serum, or of plasma.

The detection and quantification method is suitable in particular fordetecting and quantifying 25-hydroxy vitamin D₂ and/or 25-hydroxyvitamin D₃.

In step b), detection and quantification are preferably performed byperforming an immunoassay, or indeed by mass spectrometry.

The invention also provides a kit for detecting and quantifying vitaminD and/or at least one vitamin D metabolite by immunoassay and comprisinga dissociation solution of the invention. Such a kit may also include abinding partner for vitamin D or one of its metabolites and/or a solidphase on which a hapten analogous to vitamin D and/or the vitamin Dmetabolite(s) for detection is bonded, which hapten is recognized by themarked antibody.

In the context of the invention, vitamin D metabolites cover allcompounds that contain the skeleton of vitamin D₂ or the skeleton ofvitamin D₃, and in particular:

-   -   25-hydroxy vitamin D, which specifies the metabolites of vitamin        D that are hydroxylated in position 25, i.e. 25-hydroxy vitamin        D₂ and 25-hydroxy vitamin D₃; and    -   the 1,25 and 24,25-dihydroxy vitamin D forms, which specify the        metabolites of vitamin D that are dihydroxylated, respectively        in positions 1 and 25 or in positions 24 and 25.

In the methods of uses of the invention, dissociation may be performedby incorporating, in the sample for analysis, both the fluoroalkylsurfactant and also the C₁-C₄ alcohol and preferably C₁-C₃ alcohol thathave been selected, either together or separately. After suchincorporation, no separation is needed, and detection can be performeddirectly on the resulting sample. Preferably, in order to limitmanipulations, a previously prepared solution containing the fluoroalkylsurfactant and the C₁-C₄ alcohol and preferably C₁-C₃ alcohol that havebeen selected is incorporated in the sample, and in particular asolution in accordance with the invention.

In order to enhance dissociation, the sample containing the fluoroalkylsurfactant and in particular the perfluoroalkyl surfactant and C₁-C₄alcohol and preferably C₁-C₃ alcohol that have been selected issubjected to mixing. Such mixing may be performed with any appropriatedevice, and in particular by means of a reaction cone acting as apipette, as in the Applicant's Vidas® technology (Vidas® ManuelInstrument, 2005, Chapitre 2 Description fonctionnelle, 2-1 to 2-16,bioMérieux France [Vidas® Instrument manual, 2005, Chapter 2, Functionaldescription, 2-1 to 2-16, bioMérieux France]).

The vitamin D or one of its metabolites may be detected using anytechnique known to the person skilled in the art, and in particular byperforming a test using a binding partner of the analyte to be detected,and in particular an immunological test (also known as an immunoassaytest), or indeed by mass spectrometry.

Naturally, the prefix “immuno” in the term “immunoassay”, for example,should not be considered in the present application as indicatingstrictly that the binding partner is necessarily a partner ofimmunological origin, such as an antibody or an antibody fragment. As iswell known to the person skilled in the art, this term is used morewidely to designate tests and methods in which the binding partner isnot solely a partner of immunological origin and/or nature, but mayconsist, for example, of a receiver for the analyte that is to bedetected and/or quantified, the condition being that the binding partnerin question must be capable of binding to the looked-for analyte, andpreferably in specific manner. Thus, it is known to use the term“enzyme-linked immunoabsorbent assay (Elisa)” for assays that make useof binding partners that are not strictly speaking immunological, andthat are referred to more broadly as “ligand binding” assays, eventhough the term “immuno” is included in the acronym Elisa. For thepurposes of clarity and uniformity, the term “immuno” is used in thepresent application to cover any biological analysis using at least onebinding partner suitable for binding with the looked-for analyte and fordetecting and/or quantifying it, preferably in specific manner, evenwhen said binding partner is not strictly speaking of immunologicalnature or origin.

The immunological test is preferably competition assaying, which is aform of assaying well known to the person skilled in the art and whichis used when the analyte is a hapten. It consists in assaying thein-sample analyte, specifically vitamin D and/or at least one of itsmetabolites, by setting up competition between the analyte of the sampleand an analog of the analyte. The immunological reaction is thenrevealed by the presence of a tracer.

The analyte analog may be used in the competition reaction without priorcoupling or after coupling to a marker in order to form a conjugate ortracer.

Immunological assay by competition also requires the use of a bindingpartner of the analyte relative to which the analyte analog and theanalyte enter into competition. When the analyte analog is not coupledto a marker (it is not the tracer but the capture partner), the bindingpartner is marked in order to constitute the reaction tracer. When theanalyte analog is coupled to a marker (it is then the tracer), thebinding partner then becomes the capture partner.

The measured signal as emitted by the tracer is then inverselyproportional to the quantity of analyte in the sample.

The term “marker” is used to mean any molecule containing a group thatreacts with the capture partner or the analyte analog, depending on theformat, directly without chemical modification, or after chemicalmodification to include such a group, which molecule is capable ofgenerating a detectable signal either directly or indirectly. Such areactive group may in particular be a primary amine. A non-limiting listof such direct detection markers is as follows:

-   -   enzymes that produce a signal that is detectable, e.g. by        colorimetry, fluorescence, luminescence, such as horseradish        peroxydase, alkaline phosphatase, β-galactosidase, and        glucose-6-phosphate dehydrogenase;    -   chromophores such as fluorescent, luminescent, and dye        compounds;    -   radioactive molecules such as ³²P, ³⁵S, or ¹²⁵I;    -   fluorescent molecules such as Alexa or phycocyanines; and    -   electrochemiluminescent salts such as organo-metallic        derivatives based on acridinium or ruthenium.

Indirect detection systems may also be used, such as for example ligandscapable of reacting with an anti-ligand. The ligand then corresponds tothe marker for acting with the analyte analog or the binding partner toconstitute the tracer.

Ligand/anti-ligand pairs are well known to the person skilled in the artas applies for example to the following pairs: biotin/streptavidin,hapten/antibody, antigen/antibody, peptite/antibody, sugar/lectin,polynucleotide/polynucleotide complement.

The anti-ligand can then be detected directly by the above-describeddirect detection markers or may itself be detectable by using some otherligand/anti-ligand pair, and so on.

Under certain conditions, these indirect detection systems can lead tothe signal being amplified. This signal amplification technique is wellknown to the person skilled in the art, and reference may be made toprior patent applications FR 2 781 802 or WO 95/08000 in the name of theApplicant.

Depending on the type of marker used, the person skilled in the art addsreagents enabling the marking to be viewed or a signal to be emittedthat is detectable by any appropriate type of measuring apparatus, suchas for example: a spectrophotometer; a spectrofluorometer; or indeed ahigh definition camera.

The term “binding partner” for vitamin D or one of its metabolites, orindeed for a plurality of these compounds, is used to mean any moleculecapable of binding with vitamin D or one of its metabolites, or indeedwith several of these compounds (referred to in general manner as“vitamin D analytes”). As an example of a vitamin D analyte bindingpartner, mention may be made of antibodies, antibody fractions,nanofitins, vitamin D analyte receivers, or any other protein that isknown to interact with vitamin D analyte.

By way of example, the binding partner antibodies may be eitherpolyclonal antibodies or monoclonal antibodies.

Polyclonal antibodies may be obtained by immunizing an animal with thetarget vitamin D analyte as the immunogen, followed by recovering thelooked-for antibodies in purified form by taking serum from said animal,and separating said antibodies from the other constituents of the serum,in particular by affinity chromatography on a column having fixedthereon an antigen that is specifically recognized by the antibodies, inparticular the immunogen.

Monoclonal antibodies may be obtained by the hybridoma technique that iswell known to the person skilled in the art. Monoclonal antibodies mayalso be recombinant antibodies obtained by genetic engineering, usingtechniques well known to the person skilled in the art.

As examples of antibody fragments, mention may be made of Fab, Fab′,F(ab′)2 fragments and of single chain variable fragments (scFv) anddouble-stranded variable fragments (dsFv). These functional fragmentsmay be obtained in particular by genetic engineering.

Nanofitins (trade name) are small proteins that, like antibodies, arecapable of binding to a biological target, thus making it possible todetect it, capture it, or merely to target it within an organism.

The binding partners used may be specific or non-specific to the vitaminD analyte. They are said to be “specific” when they are capable ofbinding in exclusive or almost exclusive manner with the vitamin Danalyte. They are said to be “non-specific” when the selectivity of thebinding with the vitamin D analyte is weak so they are also capable ofbinding with other ligands, such as other proteins or antibodies. In apreferred implementation, specific binding partners are preferred.

Anti-vitamin D analyte antibodies are known, and they are described inparticular in Hollis, Clin. Chem. 31/11, 1815-1819 (1985) and Holis,Clin. Chem. 39/3, 529-533 (1993) and in patent EP 1 931 711. They mayalso be obtained from various suppliers such as Bioventix (UK).

Binding partners or vitamin D analogs, when they are used in capture,may optionally be bound to a medium, such as microtitration plates,latexes, reaction cones, beads having a diameter of the order of onehundred micrometers to a nanometer, using any technique well known tothe person skilled in the art. The vitamin D analogues are preferablyimmobilized on a solid phase.

It is possible in particular to make use of a medium that has beenfunctionalized with avidin and/or streptavidin and to bind on the solidphase a biotinylated analyte analog. Functionalization techniques arewell known to the person skilled in the art who can make referencethereto.

In conventional manner, in order to determine the quantity of vitamin Dand/or of at least one of its metabolites, the signal, which isinversely proportional to the quantity of analyte in the sample, may becompared with a calibration curve previously obtained using techniquesthat are well known to the person skilled in the art. Thus, by way ofexample, the calibration curve may be obtained by performing animmunological assay using the same binding partner together withincreasing known quantities of vitamin D. A curve is thus obtained withconcentration of vitamin D plotted along the abscissa axis and thecorresponding signal obtained after immunological assay plotted up theordinate axis.

The detection/quantification method of the invention may be applieddirectly to the format of commercial tests available fordetecting/quantifying vitamin D. Such formats for assaying vitamin Dand/or one of its metabolites are sold in particular by Abbott(Architect 25-OH vitamin D, ref. 3L52), DiaSorin (Liaison® 25-OH vitaminD total assay, ref. 310600), IDS (IDS-iSYS 25-hydroxy vitamin D assay,ref. IS-2700), Siemens (Advia Centaur® vitamin D total, ref. 10491994),Roche (Elecys vitamin D total).

In conventional manner, performing an immunoassay thus requires thereagents necessary for immunological detection as described above, whichreagents are to be incorporated in the sample. Advantageously, thedissociation and thus the incorporation in the sample for study, both ofthe fluoroalkyl surfactant and of the C₁-C₄ alcohol, preferably a C₁-C₃alcohol, that have been selected, are performed before incorporatingsuch reagents.

It is also possible to use mass spectrometry for performing thedetection/quantification step once the dissociation has been obtained.This technique is an analysis technique that makes it possible todetermine the molar masses of the compounds being analyzed, and alsoenables their molecular structure to be identified, and even enablesthem to be quantified. When applied to a complex mixture such as abiological fluid, it needs to be coupled to a separation technique thatenables the complexity of the fluid to be reduced. Usually thatcomprises gas chromatography (GC) or liquid chromatography (LC). Tandemmass spectrometry (MS/MS) combines two analyzers and can be used fordetection/quantification purposes. The ionic compounds selected in thefirst analyzer are analyzed more finely in the second. Such doubleanalysis serves to increase significantly the specificity of the method.For this technique, reference may be made in particular to Van den Broeket al., J. Chromatogr. B 929 161-179 (2013).

The biological sample with which the method of the invention can beperformed is any animal and preferably human biological sample thatmight contain the analyte (vitamin D or one of its metabolites), inwhich an immunoassay or a mass spectrometry analysis can be performed.Such samples are well known to the person skilled in the art. The sampleused in the assay method may optionally be modified prior to being used.As examples of such samples that are not previously modified, mentionmay be made of biological fluids such as total blood, and as examples ofsamples that have been previously modified, also known as samplederivatives, mention may be made of serum, plasma, cells recovered froma biopsy, or from a surgery, and then cultured in vitro. Theconcentration of vitamin D or of one of its metabolites may then beassayed in the culture supernatant, or indeed in the cellular lysate.

The examples below serve to illustrate the invention, but they have nolimiting character.

For each experimentally tested condition, the tables given in theexamples give the relative fluorescence value (RFV) signal as determinedby the Vidas® machine (bioMérieux). Often, a plurality of independentmeasurements were taken for each of the conditions. The “mean RFV”corresponds to the arithmetic mean of such independent measurements.

In order to verify the effectiveness of the dissociation solutions, theresults obtained with two biological samples having differentconcentrations of 25-OH vitamin D were compared by calculating the %ratio between the two signals obtained (written RFV_(No.2)/RFV_(No.1),sample No. 1 being the sample with the lower concentration of 25-OHvitamin D). For repeated independent measurements, the ratio wascalculated from mean RFVs. The smaller this ratio, the better thedissociation.

The coefficient of variation (CV) is defined as the ratio between thestandard deviation and the mean. It is often expressed as a percentage(CV %). The CV % is a measure of relative dispersion and it reflects thereproducibility of the results. A reduction in the value obtained isindicative of an improvement in reproducibility.

Some of the experiments described form part of experiment optimizationplans that were constructed by using Tagushi tables. When the optimum isa nominal value, as for a reproducibility study (fixed signal value),variance around the value may be considered as being the result of noisefactors, and thus as being detrimental to reproducibility. On the basisof repeated experimental data, the signal-to-noise ratio (S/N) may becalculated and it is defined using the formula 10×log₁₀ (mean²/standarddeviation²). The S/N ratio, sometimes known as the Taguchi constant, isindicative of the reproducibility of the results. An increase in thevalue obtained is indicative of an improvement in reproducibility.

The ratio B/B0% is the signal obtained for the tested range pointdivided by the signal obtained for the range point having 0 nanogramsper millimeter (ng/mL) of analyte, multiplied by 100.

In the various tables that follow, with the exception of Table 5, theperfluoroalkyl acids used were liquid, their percentages being given byvolume relative to the total volume of the dissociation solution. Forthe alcohol used, in all cases, the percentages are given by volumerelative to the total volume of the dissociation solution.

EXAMPLE 1—ADVANTAGE OF DISSOCIATION USING A METHANOL ANDPERFLUOROHEXANOIC ACID MIXTURE COMPARED WITH PERFLUOROHEXANOIC ACIDALONE

Preparation of Dissociation Solutions

Comparative dissociation solution: the components for preparing the PBSbuffer (5 mM of sodium hydrogen phosphate (Na₂HPO₄), 1.5 mM of potassiumdihydrogen phosphate (KH₂PO₄), 131 mM of NaCl) and 0.75%perfluorohexanoic acid were dissolved in demineralized water by stirringfor about 30 minutes (min). The pH was adjusted to 7.2 using 6N NaOH.

Solution of the invention: the components for preparing the PBS buffer(5 mM of sodium hydrogen phosphate (Na₂HPO₄), 1.5 mM of potassiumdihydrogen phosphate (KH₂PO₄), 131 mM of NaCl), 0.75% perfluorohexanoicacid, and 5% of methanol were dissolved in demineralized water bystirring for about 30 min. The pH was adjusted to 7.2 using 6N NaOH.

Method of Quantifying Total 25-OH Vitamin D

Immunological assays were performed using a Vidas® immunoanalysismachine (from bioMérieux). The single-use cone serves both as the solidphase for the reaction and as a pipetting system. The cartridge was madeup of ten wells covered in a sealed and labeled aluminum sheet. Thefirst well had a cut-out portion to facilitate inserting the sample. Thelast well was an optical cuvette in which the fluorescence of thesubstrate was measured. The various reagents needed for analysis werecontained in the intermediate wells. All of the steps of the test wereperformed automatically by the instrument. They were constituted by asuccession of suck/blow cycles of the reaction medium.

a) Sensitizing and Passivating the Cone

The cones were sensitized with 300 microliters (μL) of a carrieranti-protein antibody solution diluted to 10 micrograms per milliliter(μg/mL) in a 50 mM MES buffer of pH 6.1. After 6 hours (h) of incubationat +18/25° C., washing was performed with a 9 grams per liter (g/L)solution of NaCl. Thereafter, there were added 300 μL of a solution ofvitamin D coupled to the carrier protein and diluted to 150 nanogramsper milliliter (ng/mL) in a 200 mM tris buffer of pH 6.2 containinghuman albumin. Sensitization/passivation continued at +18/25° C.overnight. The cones were emptied, dried, and then stored at +4° C.while sheltered from moisture until they were used.

b) Pretreatment of the Sample

The sample for assay (100 μL) was introduced into the first well of thecartridge. The sample and the pretreatment reagent (comparativedissociation solution or solution of the invention) were put togetherfor separating the vitamin D contained in the sample from its bindingprotein. The Vidas® machine mixed 48 μL of sample with 340 μL ofdissociation solution; the mixture was incubated at 37° C. for 5 min.

c) Immunoassay Reaction Procedure

The pretreated sample (about 0.9 volumes) was transferred into the wellcontaining 1 volume of an anti-vitamin D antibody marked with alkalinephosphatase (conjugated, bioMérieux). The alkaline phosphatase antibodyconjugate was diluted beforehand to about 10 μg/mL in 100 mM tris bufferof pH 7.1, 300 mM NaCl, containing human albumin. The sample/conjugatemixture was incubated in the well for about 5-7 min. Thereafter thesample/conjugate mixture was incubated in the cone for an additional 5-7min approximately, during which competition took place between theantigen present in the sample and the vitamin D antigen fixed on thecone for sites of the antibody specific to conjugated anti-vitamin D.Thereafter, three successive washes with 200 mM tris buffer of pH 8.4,300 mM NaCl, Tween® 20 0.2%, were performed in order to eliminate thenon-fixed compound. During the final development step, the4-methylombelliferyl phosphate substrate was sucked out and thendelivered into the cone; the enzyme of the conjugate catalyzes thereaction of hydrolyzing the substrate into 4-methylombelliferyl, and thefluorescence it emits was measured at 450 nanometers (nm). The value ofthe fluorescence signal is inversely proportional to the concentrationof the antigen present in the sample.

Table 1 below summarizes the fluorescence signals (RFV) determined bythe Vidas® machine as a function of the dissociation solution used,using three different solutions of human serum. For each experimentalsetup, four independent measurements were made.

TABLE 1 0.75% perfluorohexanoic 0.75% acid WITHOUT methanolperfluorohexanoic acid Comparative dissociation 5% methanol solutionSolution of the invention sample No. 1 at 11 ng/mL RFV signal 3536 38183457 3674 3665 3889 3356 3922 Mean RFV 3504 3826 CV % 4% 3% S/B ratio   28.58    30.82 sample No. 2 at 20 ng/mL RFV signal 2863 3430 32903189 3496 3316 3072 3149 Mean RFV 3180 3271 CV % 9% 4% S/B ratio   21.32    28.17 % ratio 91%  85%  RFV_(No.2)/RFV_(No.1) sample No. 3at 36 ng/mL RFV signal 2642 2420 2440 2349 2761 2490 2815 2433 Mean RFV2655 2423 CV % 6% 2% S/B ratio    24.1    32.43 % ratio 76%  63% RFV_(No. 3)/RFV_(No.1)

It can be seen that adding alcohol improves dissociation (the signalratio in % decreases), while improving reproducibility (increase in thesignal-to-noise ratio and decrease in CV %).

EXAMPLE 2—COMPARING DIFFERENT ALCOHOLS

The dissociation solutions used in this example were prepared using theprocedure explained for Example 1, in a PBS buffer with pH of 7.2. Thenatures and the concentrations of the fluoroalkyl surfactant and of thealcohol were varied and are set out in Tables 2 and 3.

Otherwise, the procedure was as in Example 1.

TABLE 2 Use of perfluorohexanoic acid sample No. 1 sample No. 2 at 11ng/mL at 35 ng/mL Comparative dissociation solution: PBS + 1%perfluorohexanoic acid without alcohol Mean RFV 3224 2648 % ratioRFV_(No.2)/RFV_(No.1) — 82% Dissociation solution of the invention:PBS + 1% perfluorohexanoic acid + 5% methanol Mean RFV 3118 2323 % ratioRFV_(No.2)/RFV_(No.1) — 75% Dissociation solution of the invention:PBS + 1% perfluorohexanoic acid + 5% ethanol Mean RFV 2707 1925 % ratioRFV_(No.2)/RFV_(No.1) — 71% Dissociation solution of the invention:PBS + 1% perfluorohexanoic acid + 5% isopropanol Mean RFV 2633 2003 %ratio RFV_(No.2)/RFV_(No.1) — 76%

It can be seen that adding alcohol, regardless of the alcohol, leads toan improvement in dissociation (the signal ratio in % decreases).

TABLE 3 The use of perfluoroctanoic acid sample No. 1 sample No. 2 at 11ng/mL at 35 ng/mL Comparative dissociation solution: PBS + 0.75%perfluoroctanoic acid without alcohol Mean RFV 2684 1197  % ratioRFV_(No.2)/RFV_(No.1) — 45% Dissociation solution: PBS + 0.75%perfluorooctanoic acid + 5% methanol Mean RFV 2574 787 % ratioRFV_(No.2)/RFV_(No.1) — 31% Dissociation solution: PBS + 0.75%perfluorooctanoic acid + 5% ethanol Mean RFV 2612 759 % ratioRFV_(No.2)/RFV_(No.1) — 29%

It can be seen that adding alcohol, regardless of the alcohol, leads toan improvement in the dissociation (signal ratio in % decreases).

EXAMPLE 3—INFLUENCE OF PERFLUOROALKYL ACID Concentration

The dissociation solutions used in this heat exchanger were preparedusing the procedure explained for Example 1, in a PBS buffer with pH of7.2.

Otherwise the procedure was as in Example 1.

TABLE 4 1.25% 1.6% perfluorohexanoic acid + perfluorohexanoic acid + 2%perfluorohexanoic 5% methanol 5% methanol acid + 5% methanol samplesample sample sample sample sample No. 1 No. 2 No. 1 No. 2 No. 1 No. 2at 15 ng/mL at 35 ng/mL at 15 ng/mL at 35 ng/mL at 15 ng/mL at 35 ng/mLMean RFV 4724 3844 4054 2885 3352 2252 % ratio — 81% — 71% — 67%RFV_(No.2)/RFV_(No.1) 1.25% 1.6% perfluorohexanoic acidperfluorohexanoic acid 2% perfluorohexanoic without methanol withoutmethanol acid without methanol sample sample sample sample sample sampleNo. 1 No. 2 No. 1 No. 2 No. 1 No. 2 at 15 ng/mL at 35 ng/mL at 15 ng/mLat 35 ng/mL at 15 ng/mL at 35 ng/mL Mean RFV 3896 2589 3245 1802 25641335 % ratio — 66% — 56% — 52% RFV_(No.2)/RFV_(No.1)

It can be seen that the dissociation increases in the presence ofalcohol under all circumstances. It can also be seen that by increasingthe concentration of perfluoroalkyl acid, the dissociation is increasedfurther.

EXAMPLE 4—INFLUENCE OF ALCOHOL CONCENTRATION

The dissociation solutions used in this example were prepared using theprocedure explained for Example 1, in a PBS buffer with pH of 7.2. Thenatures and concentrations of the fluoroalkyl surfactant and of thealcohol were varied and they are set out in Table 5. Since ammoniumperfluorooctanoate is solid, the percentages associated therewith aregiven by weight of ammonium perfluorooctanoate relative to the totalvolume of the solution.

Otherwise, the procedure was as in Example 1.

TABLE 5 0.5% ammonium 0.5% ammonium 0.5% ammonium perfluorooctanoateperfluorooctanoate + perfluorooctanoate + without methanol 5% methanol10% methanol sample sample sample sample sample No. 1 No. 2 sample^(o) 1No. 2 No. 1 No. 2 at 15 ng/mL at 35 ng/mL at 15 ng/mL at 35 ng/mL at 15ng/mL at 35 ng/mL Mean RFV 2932 2341 3944 2765 4512 2697 CV % 4%  3% 4% 1% 2%  3% S/B ratio    27.37    29.77    28.62    38.04    35.85   29.92 % ratio — 80% — 70% — 60% RFV_(No.2)/RFV_(No.1)

It can be seen that dissociation increases with increasing concentrationof methanol, sometimes to the detriment of reproducibility, if theconcentration is too great. The methanol concentration should thereforebe adjusted by the person skilled in the art so as to find a compromisebetween the quantity of perfluoroalkyl surfactant and alcohol.

EXAMPLE 5—ADDING PLURONIC® F-127

The dissociation solution of the invention used in this example wasprepared using the procedure explained for Example 1, in a 50 mM Trisbuffer with pH of 7.5, with the exception that the fluoroalkylsurfactant was 1.5% perfluorohexanoic acid and the alcohol was 5%methanol. The Pluronic® F-127 was used at a concentration of 0.25%, orelse none was used. The results are given in Table 6 below.

Otherwise the procedure was as in Example 1.

TABLE 6 Without Pluronic F-127 0.25% Pluronic F-127 Mean RFV Mean RFV CV% CV % 0 ng/mL 5388   5155.5 point 1% 0% 11 ng/mL 3593 3722 point 5% 2%19 ng/mL 2693 3063 point 3% 2% 30 ng/mL 1492 2024 point 3% 0% 102 ng/mL 295  413 point 6% 2% sample 1069 1597 No. 1 3% 2% sample 2179 2914 No.2 4% 2% sample  188  401 No. 3 1% 1% sample  625 1328 No. 4 6% 2%

Adding an additional surfactant such as Pluronic® F-127 serves toimprove reproducibility (reduction in CV %). The improvement inreproducibility is greater for samples having a high concentration ofvitamin D.

EXAMPLE 6—INFLUENCE OF THE BUFFER USED

The comparative dissociation solution and the dissociation solution ofthe invention with the mention PBS as used in this example were preparedusing the procedure explained for Example 1 in a PBS buffer with pH of7.2. The natures and the concentrations of the fluoroalkyl surfactantand of the alcohol, if any, are given in Table 7 (comparative solution)and in Table 9 (solution of the invention).

The comparative dissociation solution and the dissociation solution ofthe invention bearing the mention Tris contained 50 mM of Tris, thefluoroalkyl surfactant, with or without alcohol. The components weredissolved in demineralized water by stirring for about 30 min. The pHwas adjusted to 7.5 with 6N NaOH. The natures and the concentrations ofthe fluoroalkyl surfactant and of the alcohol, if any, are given inTable 8 (comparative solution) and in Table 9 (solution of theinvention).

TABLE 7 Comparative dissociation solution: PBS + 1.5% perfluorohexanoicacid sample No. 1 sample No. 2 at 11 ng/mL at 30 ng/mL Mean RFV 25101509 % ratio RFV_(No.2)/RFV_(No.1) — 60%

TABLE 8 Comparative dissociation solution: Tris + 1.5% perfluorohexanoicacid sample No. 1 sample No. 2 at 11 ng/mL at 30 ng/mL Mean RFV 27791618 % ratio RFV_(No.2)/RFV_(No.1) — 58%

TABLE 9 Dissociation solution of the invention: PBS buffer or Trisbuffer + 1.5% perfluorohexanoic acid + 5% methanol sample No. 1 sampleNo. 2 at 11 ng/mL at 30 ng/mL Buffer PBS TRIS PBS TRIS Mean RFV 41814874 1665 2030 % ratio RFV_(No.2)/RFV_(No.1) — — 40% 42%

It can be seen that the nature of the buffer has no significantinfluence on the RFV ratio, and thus on the resulting dissociation.

The sole FIGURE plots the B/B0% ratio obtained over an ng/mL range ofanalyte for both buffers (PBS buffer and Tris buffer). The B/B0% ratiois the signal obtained for the range point under test divided by thesignal obtained for the range point having 0 ng/mL of analyte,multiplied by 100.

It can be seen that the choice of buffer used has no significantinfluence on the results obtained, whether in terms of dissociation orin terms of reproducibility.

The invention claimed is:
 1. A method of dissociation of vitamin Dand/or a vitamin D metabolite from vitamin D binding protein comprising:incorporating 5 to 10 volume of a dissociation solution comprising afluoroalkyl surfactant and an alcohol having 1 to 4 carbon atoms intoone volume of a liquid sample comprising the vitamin D and/or thevitamin D metabolite associated with the vitamin D binding protein, theincorporation of the dissociation solution resulting in the dissociationof the vitamin D and/or the vitamin D metabolite from the vitamin Dbinding protein, said dissociation solution comprising a percentage offluoroalkyl surfactant by volume when the fluoroalkyl surfactant isliquid, or by weight when the fluoroalkyl surfactant is solid, relativeto the total volume of the dissociation solution lying in the range of0.1% to 3%, and a weight percentage by volume of alcohol relative to thetotal volume of the dissociation solution lying in the range of 2% to10%, wherein the fluoroalkyl surfactant is selected from the groupconsisting of perfluorohexanoic acid, perfluoroheptanoic acid,perfluorooctanoic acid, and their salts.
 2. The method according toclaim 1, characterized in that the fluoroalkyl surfactant and thealcohol are used in quantities such that the ratio multiplied by 100 ofthe weight of surfactant, when it is solid, or of the volume ofsurfactant, when it is liquid, over the volume of alcohol lies in therange 10% to 60%.
 3. The method according to claim 1, characterized inthat the alcohol has 1 to 3 carbon atoms and is selected from the groupconsisting of methanol, ethanol, n-propanol, and isopropanol.
 4. Themethod according to claim 1, characterized in that perfluorohexanoicacid is used with methanol as the alcohol.
 5. The method according toclaim 1, characterized in that an additional surfactant is alsoincorporated into the dissociation solution, the additional surfactantis selected from the group consisting of polysorbates, polyethyleneglycol ethers, and block copolymers based on ethylene oxide andpropylene oxide.
 6. The method according to claim 1, wherein the vitaminD is 25-hydroxy vitamin D₂ and/or 25-hydroxy vitamin D₃.
 7. The methodaccording to claim 1, characterized in that the sample is a sample ofblood, of serum, or of plasma.
 8. The method according to claim 1,characterized in that the incorporation of the dissociation solution isperformed by mixing the sample with the dissociation solution comprisingthe at least one fluoroalkyl surfactant and the at least one alcoholhaving 1 to 4 carbon atoms.
 9. The method according to claim 8,characterized in that the dissociation solution further contains anadditional surfactant, the additional surfactant is selected from thegroup consisting of polysorbates, polyethylene glycol ethers, and blockcopolymers based on ethylene oxide and propylene oxide.
 10. The methodaccording to claim 8, characterized in that the dissociation solution isa buffer solution.
 11. The method according to claim 10, characterizedin that the dissociation solution is buffered to a pH lying in the range6 to
 8. 12. The method according to claim 8, characterized in that thedissociation solution does not contain any of the following compounds:dimethyl sulfoxide, dimethylformamide, N,N-dimethylacetamide,tetramethylurea, N-methylpyrrolidone,1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone, and triamidehexamethyl phosphoric acid.